The authors acknowledge W. J. Looney for carefully reviewing the manuscript.

Standard 2 des Normes internationales pour la tuberculose Etats soins que tous les patients suspects de tuberculose pulmonaire doivent soumettre au moins deux échantillons d&#39;expectoration pour examen bactériologique. Lorsque cela est possible, au moins un début de matinée échantillon doit être obtenu, comme des crachats recueillis à cette époque a le rendement le plus élevé. 11 Le Xpert MTB / RIF dosage peut être appliquée à des échantillons de crachats ou dans les sédiments de crachats concentrés préparés à partir de crachats induits ou expectoré qui sont . soit bacilles acido-résistants (BAAR) à frottis positif ou négatif de 6, 12, 13 L&#39;essai est conçu pour être utilisé avec des échantillons provenant de patients pour lesquels il est de suspicion clinique de tuberculose pulmonaire qui ont: 1) pas reçu un traitement anti-tuberculeux, 2 ) avait moins de 7 jours de traitement, ou 3) n&#39;ont pas reçu un traitement dans les 60 derniers jours. Notez qu&#39;il ne doit pas être utilisé pour surveiller les effets de 7 pharmacothérapie parce que l&#39;ADN bactérien peut persister après le traitement antimicrobien. <pclass = &quot;jove_content&quot;&gt; Traiter des spécimens cliniques, y compris les cartouches utilisées, comme s&#39;ils étaient capables de transmettre des agents infectieux. Utilisez les précautions nécessaires, comme le port des gants de protection jetables, blouses de laboratoire et des lunettes de protection, et suivre les procédures de sécurité de votre institution et des lignes directrices. 1. Les échantillons expectorations Note: Le processus que comme de nombreux spécimens à la fois, car il ya GeneXpert système DX modules disponibles pour exécuter le test! <ol><li> Étiquetez chaque Xpert MTB / RIF cartouche avec l&#39;ID de l&#39;échantillon correspondant. </li><li> Transférer 1,0 ml expectoré crachats à une conique, avec bouchon à vis tube à l&#39;aide d&#39;une pipette de transfert stérile. En variante, le spécimen peut être traité dans l&#39;original étanche récipient de collecte des expectorations. </li><li> Ajouter 2,0 ml de réactif Xpert échantillon MTB / RIF (2:1, v / v) à l&#39;expectorations aide d&#39;une pipette de transfert stérile. </li><li> Replacez le couvercle et secouer vigoureusement le tube 10-20 Fois. Sinon, le mélange peut être vortex (30 sec). </li><li> Laisser le tube de se tenir debout pendant 5 min à température ambiante. </li><li> Agiter vigoureusement le tube 10-20 fois. Vous pouvez également utiliser un vortex (30 sec). </li><li> Laisser le tube de se tenir debout pendant 10 min à température ambiante. </li><li> Inspectez les spécimens: Les échantillons doivent être liquéfié sans grumeaux visibles de crachats. </li></ol> 2. Les sédiments concentrés Note: Le processus que comme de nombreux spécimens à la fois qu&#39;il ya de GeneXpert système DX modules disponibles pour exécuter les tests! <ol><li> Étiquetez chaque Xpert MTB / RIF cartouche avec l&#39;ID de l&#39;échantillon correspondant. </li><li> Transfert de 1,5 ml de réactif Xpert échantillon MTB / RIF à une conique, avec bouchon à vis tube à l&#39;aide d&#39;une pipette de transfert stérile. </li><li> Ajouter au moins 0,5 ml sédiments concentrée sur le tube de réactif contenant l&#39;échantillon à l&#39;aide d&#39;une pipette de transfert stérile. </li><li> Replacez le couvercle et secouer le tube de vigorously 10-20 fois. Vous pouvez également utiliser un vortex (30 sec). </li><li> Laisser le tube de se tenir debout pendant 5 min à température ambiante. </li><li> Agiter vigoureusement le tube 10-20 fois. Vous pouvez également utiliser un vortex (30 sec). </li><li> Laisser le tube de se tenir debout pendant 10 min à température ambiante. </li><li> Inspectez les spécimens: Les échantillons doivent être liquéfié sans grumeaux visibles de crachats. </li></ol> 3. Chargement de la Xpert MTB / RIF Cartouche Remarque: Commencer le test dans les 30 minutes de l&#39;ajout de l&#39;échantillon de la cartouche! <ol><li> Vérifiez les étiquettes sur le VTT Xpert / FRR cartouche et l&#39;ID de l&#39;échantillon. </li><li> Ouvrez le couvercle de la cartouche. </li><li> Utilisation de la pipette de transfert stérile fourni, aspirer l&#39;échantillon liquéfié dans la pipette de transfert jusqu&#39;à ce que le ménisque est au-dessus de la marque minimum et transférer l&#39;échantillon dans l&#39;orifice ouvert de la Xpert MTB / RIF cartouche. </li></ol> Note:Distribuer lentement afin de minimiser le risque de formation d&#39;aérosols! <ol start="4"><li> Fermez le couvercle de la cartouche. </li></ol> 4. Démarrage du test Remarque: Avant de commencer le test, s&#39;assurer que le système GeneXpert Dx est équipé du logiciel GX2.1, et le Xpert MTB / RIF dosage est importé dans le logiciel 9! <ol><li> Allumez l&#39;ordinateur, puis allumez l&#39;instrument GeneXpert Dx. </li><li> Sur le bureau Windows, double-cliquez sur l&#39;icône de raccourci GeneXpert Dx. </li><li> Connectez-vous au logiciel du système GeneXpert Dx en utilisant votre nom d&#39;utilisateur et mot de passe. </li><li> Dans la fenêtre GeneXpert système DX, cliquez sur Créer un test. La cartouche de balayage de code à barres boîte de dialogue apparaît. </li><li> Dans la zone ID de l&#39;échantillon, scanner ou tapez l&#39;ID de l&#39;échantillon. </li><li> Scannez le code à barres sur le Xpert MTB / RIF cartouche. La fenêtre Test Créer s&#39;affiche. </li><li> Cliquez sur Démarrer le test </strong&gt;, et entrez votre mot de passe dans la boîte de dialogue qui apparaît. </li><li> Ouvrez la porte du module instrument avec la lumière clignote en vert, et de charger la cartouche. </li><li> Fermez la porte. Le test démarre et le voyant cesse de clignoter et reste constamment vert. Lorsque le test est terminé, la lumière s&#39;éteint. </li><li> Attendez jusqu&#39;à ce que le système libère le verrou de la porte à la fin de la course, puis ouvrez la porte du module et retirez la cartouche. </li><li> Éliminer les cartouches usagées dans les conteneurs de déchets appropriés conformément aux pratiques standard de votre institution. </li></ol> 5. Les résultats représentatifs Chaque Xpert MTB / RIF cartouche comprend des réactifs pour la détection de complexe MTB et la résistance RIF ainsi que d&#39;un contrôle du traitement de l&#39;échantillon (CPS) pour contrôler le traitement adéquat des bactéries cibles et de surveiller la présence de l&#39;inhibiteur (s) dans la réaction de PCR . Le contrôle Test de sonde (PCC) vérifie la réhydratation réactif, tube de PCR en remplissantla cartouche, la stabilité de l&#39;intégrité de la sonde, et un colorant. 9 Les amorces dans l&#39;Xpert MTB / RIF dosage amplifier une portion du gène rpoB de la paire contenant 81 de base &quot;noyau&quot; région. Cinq couleurs différentes fluorogènes nucléiques sondes d&#39;hybridation des acides, appelés balises moléculaires, d&#39;interroger l&#39;ensemble de la 81-pb de base 14. Chaque balise moléculaire a été conçu pour être aussi précis que cela ne se lie pas à son objectif si la séquence cible diffère de la rpoB de type sauvage séquence en aussi peu que une seule substitution nucléotidique. Depuis la fluorescence moléculaire balises seulement quand ils sont liés par leurs objectifs, les séquences rpoB dire de type sauvage, l&#39;absence de l&#39;une quelconque des cinq couleurs dans l&#39;essai de la différence entre l&#39;conservée séquence de type sauvage et des mutations dans la région du coeur qui sont associés à la résistance FRR 6. Le CPS devrait être positif dans un échantillon négatif et peut être négatif ou positif dans un échantillon positif. Le testrésultat sera &quot;non valide&quot; si la CPS n&#39;est pas détecté dans un test négatif. Avant le début de la réaction de PCR, le système GeneXpert Dx mesure le signal de fluorescence à partir des sondes pour surveiller la réhydratation bourrelet, par réaction tube de remplissage, l&#39;intégrité de la sonde et la stabilité de colorant. Le PCC passe si le signal de fluorescence des sondes répond aux critères d&#39;acceptation sont assignées. Les résultats sont interprétés par le système GeneXpert Dx de signaux de fluorescence mesurés et algorithmes de calcul embarqués et sont affichés dans la fenêtre Résultats de 9, comme indiqué ci-dessous: VTT Détecté: ADN cible VTT est détecté, les deux contrôles, la CPS et du PCC, de répondre aux critères d&#39;acceptation sont assignées. La baisse des valeurs de Ct représentent une plus forte concentration de départ de matrice d&#39;ADN; des valeurs plus élevées représentent Ct une concentration plus faible de la matrice d&#39;ADN. Dans les résultats VTT DETECTES &quot;Résistance FRR DETECTE&quot; (figure 1A), &quot;Résistance FRR non détecté&quot; ( VTT n&#39;est pas détectée: ADN cible VTT n&#39;est pas détecté, les deux contrôles, la CPS et du PCC, de répondre aux critères d&#39;acceptation assignées (Figure 2). Blancs: Présence ou absence de VTT ne peut être déterminée: la CPS ne répond pas aux critères d&#39;acceptation, c&#39;est à dire l&#39;échantillon n&#39;a pas été correctement traitées, ou PCR est inhibée (figure 3). Remarque: répéter le test avec un échantillon supplémentaire! Erreur: Une ou plusieurs des résultats du PCC a échoué (FAIL). Les deux VTT et de la CPS afficher aucun résultat. Remarque: répéter le test avec un échantillon supplémentaire! Si le PCC a adopté (PASS), l&#39;erreur est causée par une défaillance d&#39;un composant système. Figure 1: ADN cible VTT est détecté, les deux contrôles, la CPS et du PCC, de répondre aux critères d&#39;acceptation sont assignées. La baisse des valeurs de Ct représentent un départ plus élevé,concentration de matrice d&#39;ADN; des valeurs plus élevées Ct représentent une concentration plus faible de matrice d&#39;ADN. Dans les résultats VTT DETECTES &quot;Résistance FRR non détecté&quot; (figure 1A), &quot;Résistance FRR DETECTE&quot; (figure 1B), ou «FRR Résistance INDETERMINEE&quot; s&#39;affiche sur une ligne distincte. <img alt="Figure 1A" src="/files/ftp_upload/3547/3547fig1A.jpg" /> . Figure 1A VTT DÉTECTÉ; Résistance FRR pas détecté, à savoir que le type sauvage séquence d&#39;acide nucléique de la FRR 81-pb résistance-determining region (RRDR) du gène rpoB était présent 6. <a href="http://www.jove.com/files/ftp_upload/3547/3547fig1A.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img alt="Figure 1B" src="/files/ftp_upload/3547/3547fig1B.jpg" /> . La figure 1B VTT DÉTECTÉ; Résistance FRR DETECTE. Remplissez abandonner Probe B (flèche), qui couvre les positions 513 à 517 de la R<html> RDR du gène rpoB. 6 Dans l&#39;exemple, le séquençage partiel a révélé que la mutation du gène rpoB D516V a été détectée. 8 <a href="http://www.jove.com/files/ftp_upload/3547/3547fig1Blarge.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img alt="Figure 2" src="/files/ftp_upload/3547/3547fig2.jpg" /> Figure 2 ADN cible VTT n&#39;a pas été détecté;.. Les deux contrôles, la CPS et du PCC, de répondre aux critères d&#39;acceptation assignées <a href="http://www.jove.com/files/ftp_upload/3547/3547fig2large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img alt="Figure 3" src="/files/ftp_upload/3547/3547fig3.jpg" /> . Résultat de test valide la figure 3, la présence ou l&#39;absence de savoir VTT ne peut être déterminée: la CPS ne répond pas aux critères d&#39;acceptation, parce que l&#39;échantillon n&#39;a pas été correctement traitée, ou parce que la PCR a été inhibée.3547fig3large.jpg &quot;target =&quot; _blank &quot;&gt; Cliquez ici pour agrandir la figure.

<ol>
	<li>Sudre, P., Dam, t. e. n., Chan, G., C, A., Kochi, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis+in+the+present+time:+a+global+overview+of+the+tuberculosis+situation.">Tuberculosis in the present time: a global overview of the tuberculosis situation.</a> World Health Organisation Document WHO/TUB/91. 158, 1-47 (1991).</li><li>World Health Organisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+tuberculosis+control:+epidemiology,+strategy,+financing.">Global tuberculosis control: epidemiology, strategy, financing.</a> WHO report 2010. , (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The global plan to stop TB, 2006-2015. , (2006).</li><li>Morris, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+multiple+drug+resistance+in+clinical+isolates+of+Mycobacterium+tuberculosis.">Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis.</a> J. Infect. Dis. 171, 954-960 (1995).</li><li>Traore, H., Fissette, K., Bastian, I., Devleeschouwer, M., Portaels, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampicin+resistance+in+Mycobacterium+tuberculosis+isolates+from+diverse+countries+by+a+commercial+line+probe+assay+as+aninitial+indicator+of+multidrug+resistance.">Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as aninitial indicator of multidrug resistance.</a> Int. J. Tuberc. Lung Dis. 4, 481-484 (2000).</li><li>Helb, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Detection+of+Mycobacterium+tuberculosis+and+Rifampin+Resistance+by+Use+of+On-Demand+Near-Patient+Technology.">Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand Near-Patient Technology.</a> J. Clin. Microbiol. 48, 229-237 (2010).</li><li>Blakemore, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Analytical+Performance+of+the+Xpert+MTB/RIF.">Evaluation of the Analytical Performance of the Xpert MTB/RIF.</a> Assay. J. Clin. Microbiol. 48, 2495-2501 (2010).</li><li>Telenti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampicin-resistance+in+Mycobacterium+tuberculosis.">Detection of rifampicin-resistance in Mycobacterium tuberculosis.</a> Lancet. 341, 647-650 (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneXpert Dx System Operator Manual. , (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Standards for Tuberculosis Care (ISTC). , (2009).</li><li>Boehme, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Molecular+Detection+of+Tuberculosis+and+Rifampin+Resistance.">Rapid Molecular Detection of Tuberculosis and Rifampin Resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li><li>Marlowe, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Cepheid+Xpert+MTB/RIF+assay+for+the+Direct+Detection+of+Mycobacterium+tuberculosis+Complex+from+Respiratory+Specimens.">Evaluation of the Cepheid Xpert MTB/RIF assay for the Direct Detection of Mycobacterium tuberculosis Complex from Respiratory Specimens.</a> J. Clin. Microbiol. 49, 1621-1623 (2011).</li><li>El-Hajj, H. H., Marras, S. A. M., Tyagi, S., Kramer, F. R., Alland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampin+resistance+in+Mycobacterium+tuberculosis+in+a+single+tube+with+molecular+beacons.">Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons.</a> J. Clin. Microbiol. 39, 4131-4137 (2001).</li><li>Hillemann, D., Ruesch-Gerdes, S., Boehme, C., Richter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+extrapulmonary+tuberculosis+by+automated+GeneXpert+MTB/RIF+system.">Rapid molecular detection of extrapulmonary tuberculosis by automated GeneXpert MTB/RIF system.</a> J. Clin. Microbiol. 49, 1202-1205 (2011).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

The Xpert MTB/RIF assay integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of MTB and RIF resistance, either in expectorated sputum or concentrated sediments, in a single, easy to use system. The assay's analytical limit of detection was found to be 131 CFU/ml of sputum.6 The log-linear relationship between Ct values and the number of MTB cells present holds true from 102 to 107 CFU/ml of sputum. As clinically relevant MTB concentrations all fall within the linear range of the assay, the assay provides semi-quantitative estimates of the bacterial load.7 The assay requires only moderate laboratory infrastructure and training. The limit of detection lies at least two orders of magnitude lower than that of conventional microscopy.6 Specificity is excellent. Furthermore, the simultaneous detection of RIF-resistant MTB is a significant benefit in the setting of point-of-care testing in the era of drug-resistant TB.3
To date only limited published data on the assay's performance for diagnosing pulmonary TB in clinical settings are available.12; 13 The Xpert MTB/RIF assay's overall clinical sensitivity of detecting MTB in sputum of patients with suspected pulmonary TB was 97.6% when compared to culture as the reference. These were the findings of a prospective multi-center study that involved five study sites (Lima, Peru; Baku, Azerbaijan; Cape Town &amp; Durban, South Africa; Mumbai, India) and a total of 1462 patients. The overall pre-test probability of culture-confirmed TB in the study population was 50.7%. The sensitivity was 99.8% for smear- and culture-positive cases and 90.2% for smear-negative, culture-positive cases. Among HIV-positive patients with pulmonary TB, the sensitivity of the MTB/ RIF test was 93.9%, as compared with 98.4% in HIV-negative patients.12 In a setting of low TB prevalence, the overall agreement for detecting MTB was 89% when compared to culture; the sensitivity was 98% for smear-positive and 72% for smear-negative specimens, respectively, and the specificity was excellent.13 There was no significant difference in sensitivity between tests on untreated sputum and those on concentrated sediments.12 In the same study, the MTB/RIF test correctly detected RIF resistance in 209 of 211 patients (99.1% sensitivity) and in all 506 patients with RIF susceptibility (100% specificity), when sequencing results were taken into account.12 The authors found that performance for both case detection, and discrimination of RIF resistance, was similar across diverse sites, suggesting that the findings are likely to be widely applicable.12 Furthermore, concentrated sediments of respiratory specimens other than sputum, such as broncho-alveolar lavage and bronchial secretions, were tested with comparable sensitivity and specificity.13 Data on the assay's performance when testing extra-pulmonary TB is emerging.15
Global TB control urgently needs effective tools that permit rapid diagnosis of new TB cases and detection of RIF-resistant MTB in universal-access point-of-care settings.3 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to these settings, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions for the Xpert MTB/RIF test. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

<table border="1">
	<tbody>
		<tr>
			<td>Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Xpert MTB/RIF test</td>
			<td>Cepheid</td>
			<td>GXMTB/RIF-10</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sarstedt</td>
			<td>86.1172.001</td>
		</tr>
		<tr>
			<td>Conical tubes, 15 ml, 120 x 17 mm, PS</td>
			<td>Sarstedt</td>
			<td>62.553.542</td>
		</tr>
	</tbody>
</table>

diagnosing pulmonary tuberculosis with the xpert mtb/rif test

La tuberculose (TB) due à Mycobacterium tuberculosis (MTB) demeure un problème majeur de santé publique: l&#39;infection touche jusqu&#39;à un tiers de la population mondiale 1, et près de deux millions de personnes sont tuées par la tuberculose chaque année 2 L&#39;accès universel à haute qualité. , centrée sur le patient un traitement pour tous les patients tuberculeux est souligné par la Stratégie OMS Halte à la tuberculose. 3 La détection rapide des VTT dans les échantillons respiratoires et les traitements médicamenteux en fonction des résultats des tests de résistance des médicaments fiables sont une condition préalable à la mise en œuvre réussie de cette stratégie. Cependant, dans de nombreux domaines du monde, diagnostic de la tuberculose repose toujours sur insensibles, mal standardisés des méthodes de microscopie des expectorations. Dépistage de la tuberculose inefficace et l&#39;émergence et la transmission de souches de M. tuberculosis résistantes aux médicaments de plus en plus en péril les activités mondiales de lutte antituberculeuse. 2 Un diagnostic efficace de la tuberculose pulmonaire nécessite la disponibilité - à l&#39;échelle mondiale - du standardized, faciles à utiliser, robuste et des outils de diagnostic qui permettent la détection directe à la fois du complexe M. tuberculosis et la résistance aux antibiotiques clés, tels que la rifampicine (RIF). Ce dernier résultat peut servir de marqueur pour multirésistante VTT (MDR TB) et a été signalée dans&gt; 95% des isolats de TB-MR. 4, 5 La disponibilité rapide des résultats de test fiables est susceptible de se traduisent directement par la gestion des patients de son des décisions qui, en fin de compte, permet de guérir le patient et briser la chaîne de transmission de la tuberculose dans la communauté. 2 Cepheid (Sunnyvale, CA, USA) Xpert MTB / RIF dosage 6, 7 répond aux exigences décrites ci-dessus d&#39;une manière remarquable. Il s&#39;agit d&#39;un test d&#39;amplification nucléique acides pour 1) la détection de l&#39;ADN complexe M. tuberculosis dans les crachats ou dans les sédiments de crachats concentrés, et 2) la détection du Rif associées à la résistance des mutations du gène rpoB 8 Il est conçu pour une utilisation avec Cepheid GeneXpert Dx. e systèmeau intègre et automatise le traitement des échantillons, l&#39;amplification d&#39;acide nucléique, et la détection des séquences cibles en utilisant PCR en temps réel et de la transcriptase inverse PCR. Le système se compose d&#39;un instrument, un ordinateur personnel, lecteur de codes barres, et le logiciel préchargé pour l&#39;exécution des tests et l&#39;affichage des résultats. 9 Il emploie à usage unique jetables Xpert MTB / RIF cartouches qui contiennent des réactifs de PCR et d&#39;accueillir le procédé PCR. Parce que les cartouches sont autonomes, la contamination croisée entre les échantillons est éliminée. 6 actuelles nucléiques méthodes d&#39;amplification des acides utilisés pour détecter VTT sont complexes, de main-d&#39;œuvre, et techniquement exigeant. Le Xpert MTB / RIF test a le potentiel d&#39;apporter standardisé, sensible et très spécifique des tests de diagnostic pour la tuberculose et la résistance aux médicaments à l&#39;universel l&#39;accès au point de milieux de soins 3, à condition qu&#39;ils soient en mesure de se le permettre. Afin de faciliter l&#39;accès, la Fondation pour l&#39;innovation en matière de Nouveaux Diagnostics (FIND) a négocié signifiréductions de prix dévers. Les prix actuels TROUVEZ-négociés, ainsi que la liste des pays éligibles pour les rabais, sont disponibles sur le Web 10.

Watch this Scientific Journal Video about Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test at JoVE.com

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Diagnostic de la tuberculose pulmonaire avec le Xpert MTB / RIF test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

diagnosing-pulmonary-tuberculosis-with-the-xpert-mtbrif-test

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JoVE Journal

Immunology and Infection

79.8K vues.  University of Bern. The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

Vidéo: Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

La croissance de la Mycobacterium tuberculosis Les biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Recherche

Immunologie et infection

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

Mesures systolique ventriculaire droite pression en combinaison avec la récolte du poumon et des échantillons de tissus immunitaires chez des souris

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Isolement et caractérisation de neutrophiles avec des propriétés anti-tumorales

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Analyse des Lymphocytes extravasation L&#39;utilisation d&#39;un<em&gt; In vitro</em&gt; Modèle de la barrière hématoencéphalique humaine

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

Un<em&gt; In Vitro</em&gt; Ensemble de liaison de caséine qui prédit la pénétration des drogues dans les lésions tuberculeuses

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Test de tolérance au Glucose intrapéritonéal, mesure de la fonction pulmonaire et la Fixation du poumon pour étudier l’Impact de l’obésité et des troubles du métabolisme sur résultats pulmonaires

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Imagerie de Mycobacterium tuberculosis chez la souris avec la journaliste Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Co-Immunoprécipitation dosage pour l’étude des Interactions fonctionnelles entre les récepteurs et les Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Application de perturbations de massage cohérentes sur les veaux de souris et surveillance des changements de pression intramusculaire qui en résultent

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...